• Archives of Pharmacal Research
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• v.21 no.3
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• pp.310-314
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• 1998

DW-116 is a new fluoroquinolone antimicrobial agent with a broad spectrum. In order to elucidate the resistance mechanism to DW-116 in Acinetobacter spp. bacteria, total chromosomal DNA was isolated from 10 strains of Acinetobacter spp. resistant to DW-116. Quinolone resistance determinant region (QRDR) of DNA gyrase gene was amplified by PCR. The 345 bp nucleotide fragment yielded was inserted into pKF 3 which was used as the vector. Comparisons of the DNA sequences of 8 strains with that of the wild type strain revealed a Ser-83 to Leu mutation in mutants and all ten strains contained one silent mutation$(T{\rightarrow}G)$in QRDR. From Acinetobacter MB4-8 strain, DNA gyrase was isolated and purified, through novobiocin-sepharose, heparin-sepharose affinity column chromatography. The enzyme was composed of two subunits and the molecular mass of subunits A and B were 75.6 and 51.9 kDa, respectively. The supercoiling activity of the reconstituted DNA gyrase composed of subunit A from Acinetobacter MB4-8 and subunit B from E. coli was not inhibited by $128{\mu}\textrm{g}$ml of ciprofloxacin. It might be said that one of the resistance mechanisms to DW-116 in Acinetohacter MB4-8 was subunit A alteration of DNA gyrase.

• Korean Journal Plant Pathology
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• v.11 no.1
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• pp.73-79
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• 1995

Complementary DNAs (cDNAs) to the coat protein gene of an ordinary Korean strain of potato virus Y (PVY-OK) isolated from potato (cv. Superior) were synthesized and cloned into a plasmid pUC119 and sequenced. The RNA of the virus propagated in tobacco (Nicotinaa sylvestris) was extracted by the method of phenol extraction. The first strand of cDNAs to the coat protein penomic RNA of the virus was made by Moloney murine leukemia virus reverse transcriptase. The cDNA were synthesized and amplified by the method of polymerase chain reaction (PCR) using a pair of oligonucleotide primers. PVYCP3P and PVYCP3M. The size of cDNAs inserted in pUC119 plasmid was estimated as about 840 bp upon agarose gel electrophoresis. Double stranded cDNAs were transformed into the competent cell of E. coli JM109. Sequence analysis of cDNAs was conducted by the dideoxynucleotide chain termination method. Homology of cDNAs of the PVY-OK coat protein genomic RNA with those of PVY-O (Japan), PVY-T (Japan), PVY-TH (Japan), PVYN (The Netherlands),and PVYY (France) was represented as 97.3%, 88.9%, 89.3%, 89.6% and 98.5%, respectively. Homology at the amino acid level turned out to the be 97.4%, 92.5%, 92.9%, 92.9% and 98.5%, respectively.

• Mycobiology
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• v.34 no.2
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• pp.104-107
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• 2006

Flammulina velutipes was transformed efficiently by Agrobacterium-mediated transformation system. The transformation frequency was about 16% with the gill tissues of the fungal fruiting body. Southern hybridization and genetic analysis suggest that the introduced DNA was inserted onto different locations of the fungal genome, and inherited stably to the next generation via basidiospores. Transformation or gene tagging with Agrobacterium T-DNA based vector should be useful for wide ranges of genetic or molecular biological studies of the mushroom.

• BMB Reports
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• v.37 no.6
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• pp.657-662
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• 2004

Oligoribonucleotides containing 8-oxo-7,8-dihydroguanosine (8-oxoG) and 8-oxo-7,8-dihydro-2'-O-methylguanosine (8-oxoG-Me) were synthesized. The base pairing properties of 8-oxoG and 8-oxoG-Me in oligoribonucleotide in cDNA synthesis by reverse transcriptases were studied. dCMP was preferentially incorporated into the site opposite 8-oxoG or 8-oxoG-Me than into other dNMPs. TMP and dCMP were inserted preferentially into sites opposite 8-oxoG or 8-oxoG by reverse transcriptases. HIV-RT did not incorporate TMP, but RAV2-RT incorporated 50% more TMP than dCMP into the site opposite 8-oxoG. In the site opposite 8-oxoG-Me TMP was substantially incorporated by HIV-RT or RAV2-RT. Thermodynamic analysis of the DNA. RNA heteroduplex containing 8-oxoG revealed that 8-oxoG and 8-oxoG-Me formed base pairs with cytidine and thymidine with similar stability. The thermodynamic parameter (${\Delta}G^{\circ}$) demonstrated that the formation of duplexes between 8-oxoG or 8-oxoG-Me and cytidine or thymidine is more thermodynamically favorable than with adenosine and guanosine. However, differences in the melting temperature and ${\Delta}G^{\circ}$'s of 8-oxoG/dC and 8-oxoG/T were much smaller than between G/dC and G/T. CD spectra showed that DNA . RNA containing 8-oxoG or 8-oxoG-Me duplexes showed similarities between the A-type RNA and B-type DNA conformations.

• Parasites, Hosts and Diseases
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• v.46 no.4
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• pp.209-216
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• 2008

A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5'-RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.

• Parasites, Hosts and Diseases
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• v.44 no.3
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• pp.251-254
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• 2006

Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3'-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5'-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 amino acids without significant homology to any other known proteins. This sequence contains an amino terminal stop transfer sequence downstream of a short neutral sequence, hydrophilic middle sequence, and hydrophobic carboxy terminus. It is suggested that the ROP6 is inserted into the rhoptry membrane with both N- and C-termini.

• Journal of Plant Biotechnology
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• v.48 no.4
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• pp.223-227
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• 2021

Several hypotheses for the origin of cultivated sweetpotato [Ipomoea batatas L. (Lam)] have been suggested but the exact progenitor is still unknown. Based on the results of RFLP patterns, microsatellite markers, SNP markers, FISH analyses, and genome analyses of haplotypes, wild species belonging to batatas group, I. trifida, I. leucantha, I. littoralis, I. tabascana, I. tenuissima, I. tiliacea, and I. triloba have been suggested as a progenitor. However, recently, advanced genomic technologies and characterization of the inserted T-DNA fragments of Agrobacterium in the genome of cultivated sweetpotato and wild species through horizontal gene transfer suggest that there may be an older progenitor than the wild species suggested so far.

• The Korean Journal of Zoology
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• v.28 no.3
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• pp.166-178
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• 1985

In order to express hepatitis B surface antigen $(HB_sAg)$ containing pre-surface antigen region in mammalian calls, 2.7 kb DNA fragment containing pre-surface region-$HB_sAg$ gene poly(A) addition site of HBV genome was cloned into simian virus 40(SV 40) based chimeric vector pSVOB. 2.7 kb DNA fragment was derived from pHBVD 107 containing tandem copies of the HBV genome in a head-to-tail arrangement by Bgl II digestion. Construction of the vector pSVOE involved the incorporation of SV40 sequences spanning the viral origin of replication and 72 bp repeats (enhancer) into a pBR 322 derivative lacking sequences which inhibit replication in mammalian cells. Bam HI linker was inserted at the Pvu II site in the proximity of SV40 late promoter of pSVOE and named as pSVOB. To construct the recombinant plasmid pSVBS, pHBVD 107 was digested with Bgl II to isolate 2.7kb DNA fragment and the fragment was ligated into the Bam HI site of pSVOB by ligation. Preliminary result showed that the recombinant plasmid pSVBS produced $HB_sAg$ in the monkey cell producing large T antigen (COS cell).

• Biomedical Science Letters
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• v.8 no.3
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• pp.107-113
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• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.3
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• pp.217-222
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• 2004

As Agrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast, Saccharomyces cerevisiae, a variety of fungi were subjected to the A. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. The A. tumefaciens-mediated transformation of chestnut blight fungus, Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1${\times}$10$\^$6/ conidia of C. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.